MicroRNAs (miRNAs) play a crucial role in the development and progression of human cancers, including gastric cancer (GC). The discovery of miRNAs may provide a new and powerful tool for studying the mechanism, diagnosis, and treatment of GC. In this study, we aimed to investigate the role of miR-326 in the development and progression of GC. Quantitative PCR (qPCR) was used to measure the expression level of miR-326 in GC tissues and cell lines. We found that miR-326 was significantly downregulated during GC. In addition, overexpression of miR-326 inhibited GC cell proliferation. Fluorescence-activated cell sorting (FACS) further showed that miR-326 significantly induced GC cell G2/M arrest. Subsequent dual-luciferase reporter assay identified one of the proto-oncogene NOB1 as a direct target of miR-326, and NOB1 can save growth inhibition caused by miR-326. We also confirmed that the growth inhibition caused by miR-326 is associated with AKT pathway activation. Taken together, our results indicate that miR-326 could serve as a potential diagnostic biomarker and therapeutic option for GC in the near future.